scholarly journals Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells.

1996 ◽  
Vol 16 (4) ◽  
pp. 1695-1705 ◽  
Author(s):  
M Crossley ◽  
E Whitelaw ◽  
A Perkins ◽  
G Williams ◽  
Y Fujiwara ◽  
...  
Keyword(s):  
Zinc Finger Protein ◽  
Binding Protein ◽  
Globin Gene ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Isolation And Characterization ◽  
Gel Shift ◽  
In Erythroid Cells

CACCC boxes are among the critical sequences present in regulatory elements of genes expressed in erythroid cells, as well as in selected other cell types. While an erythroid cell-specific CACCC-box-binding protein, EKLF, has been shown to be required in vivo for proper expression of the adult beta-globin gene, it is dispensable for the regulation of several other globin and nonglobin erythroid cell-expressed genes. In the work described here, we searched for additional CACCC-box transcription factors that might be active in murine erythroid cells. We identified a major gel shift activity (termed BKLF), present in yolk sac and fetal liver erythroid cells, that could be distinguished from EKLF by specific antisera. Through relaxed-stringency hybridization, we obtained the cDNA encoding BKLF, a highly basic, novel zinc finger protein that is related to EKLF and other Krüppel-like members in its DNA-binding domain but unrelated elsewhere. BKLF, which is widely but not ubiquitously expressed in cell lines, is highly expressed in the midbrain region of embryonic mice and appears to correspond to the gel shift activity TEF-2, a transcriptional activator implicated in regulation of the simian virus 40 enhancer and other CACCC-box-containing regulatory elements. Because BKLF binds with high affinity and preferentially over Sp1 to many CACCC sequences of erythroid cell expressed genes, it is likely to participate in the control of many genes whose expression appears independent of the action of EKLF.

scholarly journals Antagonistic Regulation of β-Globin Gene Expression by Helix-Loop-Helix Proteins USF and TFII-I

2006 ◽  
Vol 26 (18) ◽  
pp. 6832-6843 ◽  
Author(s):  
Valerie J. Crusselle-Davis ◽  
Karen F. Vieira ◽  
Zhuo Zhou ◽  
Archana Anantharaman ◽  
Jörg Bungert
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Adult Stage ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Globin Gene Expression ◽  
In Erythroid Cells

ABSTRACT The human β-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the β-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased β-globin gene expression, while diminishing TFII-I activity increased β-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult β-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the β-globin gene locus and play important roles in restricting β-globin gene expression to the adult stage of erythropoiesis.

scholarly journals Histone Methyltransferase Setd8 Represses Gata2 Expression and Regulates Erythroid Maturation

2015 ◽  
Vol 35 (12) ◽  
pp. 2059-2072 ◽  
Author(s):  
Jeffrey Malik ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Global Gene Expression ◽  
Regulatory Elements ◽  
Histone Methyltransferase ◽  
Tissue Type ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Histone H4 ◽  
Nuclear Condensation ◽  
Erythroid Maturation ◽  
In Erythroid Cells

Setd8 is the sole histone methyltransferase in mammals capable of monomethylating histone H4 lysine 20 (H4K20me1). Setd8 is expressed at significantly higher levels in erythroid cells than any other cell or tissue type, suggesting that Setd8 has an erythroid-cell-specific function. To test this hypothesis, stable Setd8 knockdown was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, nontransformed model of erythroid maturation. Knockdown of Setd8 resulted in impaired erythroid maturation characterized by a delay in hemoglobin accumulation, larger mean cell area, persistent ckit expression, incomplete nuclear condensation, and lower rates of enucleation. Setd8 knockdown did not alter ESRE proliferation or viability or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown demonstrated that in erythroid cells, Setd8 functions primarily as a repressor. Most notably, Gata2 expression was significantly higher in knockdown cells than in control cells and Gata2 knockdown rescued some of the maturation impairments associated with Setd8 disruption. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. These results suggest that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2 expression.

scholarly journals The HMG Domain Protein SSRP1/PREIIBF Is Involved in Activation of the Human Embryonic β-Like Globin Gene

1998 ◽  
Vol 18 (5) ◽  
pp. 2617-2628 ◽  
Author(s):  
Michael A. Dyer ◽  
Patrick J. Hayes ◽  
Margaret H. Baron
Keyword(s):  
Developmental Stage ◽  
Regulatory Element ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Erythroid Cell ◽  
Expression Cloning ◽  
Erythroid Cells ◽  
Specific Expression ◽  
Binding Factor

ABSTRACT The human embryonic β-like globin (ɛ-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the ɛ-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An ∼85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human ɛ-globin gene.

scholarly journals The hydroxyurea-induced small GTP-binding protein SAR modulates γ-globin gene expression in human erythroid cells

Blood ◽  
2005 ◽  
Vol 106 (9) ◽  
pp. 3256-3263 ◽  
Author(s):  
Delia C. Tang ◽  
Jianqiong Zhu ◽  
Wenli Liu ◽  
Kyung Chin ◽  
Jun Sun ◽  
...  
Keyword(s):  
Cell Growth ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Globin Gene ◽  
K562 Cells ◽  
Pi3 Kinase ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding

AbstractHydroxyurea (HU), a drug effective in the treatment of sickle cell disease, is thought to indirectly promote fetal hemoglobin (Hb F) production by perturbing the maturation of erythroid precursors. The molecular mechanisms involved in HU-mediated regulation of γ-globin expression are currently unclear. We identified an HU-induced small guanosine triphosphate (GTP)–binding protein, secretion-associated and RAS-related (SAR) protein, in adult erythroid cells using differential display. Stable SAR expression in K562 cells increased γ-globin mRNA expression and resulted in macrocytosis. The cells appeared immature. SAR-mediated induction of γ-globin also inhibited K562 cell growth by causing arrest in G1/S, apoptosis, and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. SAR also enhanced both γ- and β-globin transcription in primary bone marrow CD34+ cells, with a greater effect on γ-globin than on β-globin. Although up-regulation of GATA-2 and p21 was observed both in SAR-expressing cells and HU-treated K562 cells, phosphatidylinositol 3 (PI3) kinase and phosphorylated ERK were inhibited specifically in SAR-expressing cells. These data reveal a novel role of SAR distinct from its previously known protein-trafficking function. We suggest that SAR may participate in both erythroid cell growth and γ-globin production by regulating PI3 kinase/extracellular protein–related kinase (ERK) and GATA-2/p21-dependent signal transduction pathways.

scholarly journals ZNF410 uniquely activates the NuRD component CHD4 to silence fetal hemoglobin expression

2020 ◽  
Author(s):  
Xianjiang Lan ◽  
Ren Ren ◽  
Ruopeng Feng ◽  
Lana C. Ly ◽  
Yemin Lan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
List Type ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Adult Type ◽  
In Erythroid Cells

SummaryMetazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA binding protein that in human erythroid cells directly and measurably activates only one gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplant settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells whose perturbation might offer new therapeutic opportunities in the treatment of hemoglobinopathies.HighlightsA CRISPR screen implicates ZNF410 in fetal globin gene repressionThe CHD4 gene is the singular direct ZNF410 target in erythroid cellsThe fetal globin genes are exquisitely sensitive to CHD4 levelsFive C2H2 zinc fingers of ZNF410 recognize the major groove of a 14 base pair sequence

scholarly journals Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

1991 ◽  
Vol 11 (3) ◽  
pp. 1239-1247 ◽  
Author(s):  
M H Baron ◽  
T Maniatis
Keyword(s):  
Transcriptional Activation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Aberrant Expression ◽  
Previous Demonstration ◽  
High Level

We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.

scholarly journals A CTCF-Dependent Silencer Located in the Differentially Methylated Area May Regulate Expression of a Housekeeping Gene Overlapping a Tissue-Specific Gene Domain

2006 ◽  
Vol 26 (5) ◽  
pp. 1589-1597 ◽  
Author(s):  
Denis Klochkov ◽  
Héctor Rincón-Arano ◽  
Elena S. Ioudinkova ◽  
Viviana Valadez-Graham ◽  
Alexey Gavrilov ◽  
...  
Keyword(s):  
Gene Transcription ◽  
Globin Gene ◽  
Housekeeping Gene ◽  
Regulatory Elements ◽  
Ctcf Binding ◽  
Specific Gene ◽  
Open Chromatin ◽  
Erythroid Cells ◽  
Tissue Specific ◽  
In Erythroid Cells

ABSTRACT The tissue-specific chicken α-globin gene domain represents one of the paradigms, in terms of its constitutively open chromatin conformation and the location of several regulatory elements within the neighboring housekeeping gene. Here, we show that an 0.2-kb DNA fragment located ∼4 kb upstream to the chicken α-globin gene cluster contains a binding site for the multifunctional protein factor CTCF and possesses silencer activity which depends on CTCF binding, as demonstrated by site-directed mutagenesis of the CTCF recognition sequence. CTCF was found to be associated with this recognition site in erythroid cells but not in lymphoid cells where the site is methylated. A functional promoter directing the transcription of the apparently housekeeping ggPRX gene was found 120 bp from the CTCF-dependent silencer. The data are discussed in terms of the hypothesis that the CTCF-dependent silencer stabilizes the level of ggPRX gene transcription in erythroid cells where the promoter of this gene may be influenced by positive cis-regulatory signals activating α-globin gene transcription.

Regulated expression of human alpha- and beta-globin genes in transient heterokaryons

1991 ◽  
Vol 11 (3) ◽  
pp. 1239-1247
Author(s):  
M H Baron ◽  
T Maniatis
Keyword(s):  
Transcriptional Activation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Aberrant Expression ◽  
Previous Demonstration ◽  
High Level

We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.

Comparing Strategies to Reactivate Fetal Globin Expression for the Treatment of Beta-Globinopathies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 333-333
Author(s):  
Laura Breda ◽  
Jeremy W. Rupon ◽  
Irene Motta ◽  
Wulan Deng ◽  
Gerd A. Blobel ◽  
...  
Keyword(s):  
Cell Viability ◽  
Zinc Finger Protein ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Loop Formation ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Chromatin Looping

Abstract The hemoglobinopathies, such as β-thalassemia and sickle cell anemia (SCA), are characterized by mutations of the β-globin gene resulting in either decreased or functionally abnormal hemoglobin (Hb) production. As bone marrow transplant is the only curative option for these patients, there is a strong need for new therapeutic approaches. Both β-thalassemia and SCA represent ideal targets for gene therapy since introduction of a normal β-globin gene can ameliorate the phenotype, as we and others have shown previously. Overcoming the developmental silencing of the fetal γ-globin gene represents an additional approach for the treatment of hemoglobinopathies. Here, we directly compare a recently established approach to activate the γ-globin gene using forced chromatin looping with pharmacologic approaches to raise γ-globin expression. The β-type globin genes are activated through dynamic interactions with a distal upstream enhancer, the locus control region (LCR). The LCR physically contacts the developmental stage appropriate globin gene via chromatin looping, a process partially dependent on the protein Ldb1. Previously, we have shown that tethering Ldb1 to the murine β-globin promoter with a custom designed zinc finger protein (ZF-Ldb1) can induce loop formation and β-globin transcription in an erythroid cell line (Deng et al., 2012). Further work showed that forced chromatin looping can be exploited to potently reactivate fetal globin gene expression in adult human erythroid cells (Deng et al., 2014). Here we compared the efficacy and toxicity of ZF-Ldb1 to pharmacologic compounds that induce HbF in cultured hematopoietic stem progenitor cell-derived erythroid cultures from normal and SCA donors. ZF-Ldb1 increased HbF synthesis in SCA erythroid cells (N=8) up to 86% and, concurrently, reduced sickle Hb (HbS) below 15%, consistent with previous studies of erythroid cells from normal probands. Preliminary results obtained from treating SCA specimens (N=3) show that the induction of HbF in cells treated with ZF-Ldb1 is twice as high (+35.55% ± 8.34%, at a dose of ~ one ZF-Ldb1 transgene copy per cell) as that observed using pomalidomide (+16.50% ± 14.57%, 20μM) and decitabine (+15.60% ± 12.36%, 0.5μM). Tranylcypromine and hydroxyurea showed the lowest HbF increase (+9.67% ± 3.26% and +5.06 ± 2.82%, 1.5μM and 150μM respectively). Importantly, decitabine and pomalidomide treatment lowered cell viability to 39% and 26%, respectively, while ZF-Ldb1 expressing cells retained normal viability similar to control populations. In related experiments, we are comparing the expression of a battery of genes known to regulate HbF levels (BCL11A, SOX6, KLF1 and C-Myb) in normal and SCA derived erythroid cells treated with ZF-Ldb1 or HbF inducers and compared to controls. Preliminary analyses indicate altered expression of KLF1 in SCA versus normal cells, consistent with a superior response of SCA cells to HbF induction. In conclusion, lentiviral-mediated ZF-Ldb1 gene transfer appears superior to pharmacologic compounds in terms of efficacy and cell viability further supporting suitability for the reactivation of HbF in SCA erythroid cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Downregulation of Transcription Factor LRF/ZBTB7A Increases Fetal Hemoglobin Expression in β-Thalassemia/Hemoglobin E Erythroid Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3549-3549
Author(s):  
Sukanya Chumchuen ◽  
Tanapat Pornsukjantra ◽  
Pinyaphat Khamphikham ◽  
Usanarat Anurathapan ◽  
Orapan Sripichai ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Hemoglobin E ◽  
In Erythroid Cells

LRF/ZBTB7A is a transcription factor that has been recently identified as a new key regulator of fetal hemoglobin (HbF; α2γ2) production in erythroid cells. Reduction of LRF/ZBTB7A expression led to increases in levels of HbF in human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroblast and in human immortalized erythroid line (HUDEP-2). Since reactivation of γ-globin gene is associated with the improvement of clinical manifestations of β-hemoglobinopathy patients, decrement in LRF/ZBTB7A expression might be a substantial interest as a novel target for gene therapy in β-thalassemia. In this study, we investigated the effects of LRF/ZBTB7A downregulation in erythroid cells derived from β-thalassemia/HbE patients in order to evaluate its therapeutic potential. The hematopoietic CD34+ progenitor cells were collected from 3 patients and 3 healthy normal individuals' peripheral blood and subjected for in vitro erythroblast culture. The cells were transduced with lentivirus carrying LRF/ZBTB7A specific shRNA, and used untransduced cells and non-targeted control shRNA (shNTC) as experimental controls. The LRF/ZBTB7A shRNA reduced LRF/ZBTB7A transcript and protein to nearly undetectable levels. Interestingly, downregulation of LRF/ZBTB7A increased expression of γ-globin, ε-globin and ζ-globin in both adult normal and β-thalassemia/HbE derived cells, whereas α-globin, β-globin and δ-globin expression were decreased. As previously reported, we found that the LRF/ZBTB7A knockdown produced a robust increase in HbF levels in both normal (43.3±9.0% vs. 5.9±2.1% in shNTC) and β-thalassemia/HbE erythroblasts (78.1±3.5% vs. 26.3±3.9% in shNTC). Noteworthy, the delay of erythroid differentiation was observed in the LRF/ZBTB7A knockdown cells of both derived from β-thalassemia/HbE patients and normal control, suggesting an additional role of LRF/ZBTB7A in regulating erythroid maturation. These data support the manipulation of LRF/ZBTB7A as one of the most interesting gene therapy candidates for treating the β-thalassemia, but the effect on erythroid cell maturation is needed to be concerned and required further investigation. Disclosures No relevant conflicts of interest to declare.

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